Assessment of wound healing activity in diabetic mice treated with a novel therapeutic combination of selenium nanoparticles and platelets rich plasma

Diabetic wound healing is sluggish, often ending in amputations. This study tested a novel, two-punch therapy in mice—Selenium nanoparticles (Se NPs) and platelet-rich plasma (PRP)—to boost healing. First, a mouse model of diabetes was created. Then, Se NPs were crafted for their impressive antioxidant and antimicrobial powers. PRP, packed with growth factors, was extracted from the mice's blood. Wound healing was tracked for 28 days through photos, scoring tools, and tissue analysis. Se NPs alone spurred healing, and PRP added extra fuel. Furthermore, when used in combination with PRP, the healing process was accelerated due to the higher concentration of growth factors in PRP. Notably, the combination of Se NPs and PRP exhibited a synergistic effect, significantly enhancing wound healing in diabetic mice. These findings hold promise for the treatment of diabetic wounds and have the potential to reduce the need for lower limb amputations associated with diabetic foot ulcers. The innovative combination therapy using Se NPs and PRP shows great potential in expediting the healing process and addressing the challenges of impaired wound healing in individuals with diabetes. This exciting finding suggests this therapy could change diabetic wound management, potentially saving limbs and improving lives.


Results
The transmission electron microscopy (TEM) analysis depicted the spherical shape of the Selenium nanoparticles (Se NPs) with an average diameter of 31.6 ± 1.3 nm (Fig. 1a).Upon the reaction of sodium selenite with ascorbic acid, the selenium underwent reduction to elemental selenium (Se 0 ), leading to a colour change from colourless to orange, indicating the occurrence of the reduction reaction and the formation of Se NPs.Se/PRP NCs show slight increase in the particle size to 35 ± 0.6 nm (Fig. 1b).The change in colour of the Se NPs dispersion was further highlighted in the UV-visible absorption spectrum (Fig. 1c), where the maximum absorbance was observed at 258 nm and the combination of Se NPs with PRP showed a shift to longer wavelength by to 273 nm where PRP showed maximum absorbance at 287 nm and 412 nm.The Fourier-transform infrared (FTIR) spectrum of Se NPs (Fig. 1d) exhibited distinct peaks at specific wavenumbers: 3438 cm −1 (OH in H 2 O), 2034 cm −1 (CN in polyvinylpyrrolidone (PVP)), 1635 cm −1 (C = O in PVP), 1116 cm −1 (amide in PVP), and 690 cm −1 (Se-O).These peaks provided valuable information about the functional groups present in the Se NPs.The purified PRP were characterized using a FTIR spectrophotometer.The hydroxyl group was absorbed as strong intensities between 3000 to 3500 cm −1 and ether signals (C-O-C) in sugar units were observed at 2800 to 2950 cm −1 .A further band at 1050 to 1150 cm −1 was due to the aliphatic hydrogen 35 .The combination between Se NPs and PRP showed new peaks at 2800-2950 cm −1 of the C-O-C in sugar units of PRP to the FTIR spectrum of Se NPs (Fig. 1d).The X-ray diffraction (XRD) pattern of the Se NPs (Fig. 1e) displayed characteristic diffraction peaks at angles of 23.63º, 30.04º, 41.23º, 44.46º, 45.71º, 52.01º, and 56.49º.These peaks corresponded to the (100), ( 101), ( 110), ( 102), ( 111), ( 200), (201), and (003) lattice planes of hexagonal Se, aligning well with the characteristic peaks in the standard card (PDF65-1876).Se/PRP NCs do not show changes in the XRD patterns.XRD is not typically used to analyse PRP due to its limitations in characterizing biological samples.PRP is a complex mixture of various components, including platelets, white blood cells, plasma proteins, and lipids.These components have varying sizes, shapes, and internal structures, making it difficult to obtain clear and interpretable XRD patterns.Se nanosuspension showed water solubility with zeta potential + 44 mV which is a stable suspension system (Fig. 1f).PRP showed zeta potential − 14 mV (Fig. 1g) where the combination between Se NPs and PRP showed + 16.8 mV (Fig. 1h).The zeta potential of + 16.8 mV for the Se NPs-PRP combination suggests limited stability according to Colorado State University's criteria and that may be due to the surface coating of Se NPs can significantly impact their zeta potential.The proteins and other components in PRP can interact with the Se NPs and influence their surface charge.This interaction could lead to a decrease in zeta potential if the interaction is mainly through negatively charged groups.The pH and ionic strength of the suspending medium can also affect the zeta potential.Lower pH or higher ionic strength can generally reduce the magnitude of the zeta potential 36 .

General observation of wound closure
To examine the therapeutic potential of Se NPs-PRP in diabetic wound healing, an alloxan-induced diabetic mouse model was used to generate type 2 diabetes in mice.The induction efficiency of diabetes with a single dose of alloxan monohydrate (150 mg/kg) was 100% and the survival rate was 100% during the experiment.After surgical wounding of all mice, wound closure was monitored and photo-documented (Fig. 2).Throughout the 21-day treatment period, the mice exhibited no negative effects, as well as none of the experimental groups experienced any appreciable changes in body weight.There was no bleeding or disturbance of the newly formed granulation tissue when all scaffolds were removed.The findings showed that in groups 1, 3, 4, 5, 6, and 7 there was not any clinical or gross sign of intercurrences in wound healing, such as edema, erythema, or suppuration.Groups 5, 6, and 7 showed accelerated wound closure onset.In group 2, the healing process was generally prolonged, incomplete, and uncoordinated until day 21st.Group 3 and 4 showed a slight improvement in healing www.nature.com/scientificreports/rate with minor scaring.Figure 3 shows the wound area concerning different days (the rate of wound closure over time).

The Bates-Jensen wound assessment tool (BWAT)
BWAT states that wound regeneration is represented by total scoring less than or equal to 13 while total scoring greater than this value means wound deterioration.To calculate this total score, wound criteria elements are summed up.Each wound criterion is assessed by numbers from 1 to 5. Number 1 refers to improvement toward closure while 5 implies impaired wound healing.From the findings in Fig. 4, cumulative BWAT score values ranged from 13 to 32.Group 2 had higher total scores than the other groups which indicates more severe wound status.All the groups scored 13 on day 21 expect group 2 which scored 27.Until day 28, group 2 scored 23 which means impaired healing process.Heat Map representing all wound criteria and scoring of Bates-Jensen Wound Assessment Tool in groups 1:7 group 1 normal untreated mice, group 2; diabetic untreated mice, group 3; diabetic mice treated with PRP, group 4; diabetic mice treated with Se NPs, group 5; diabetic mice treated with Se NPs + PRP (1:1), group 6 Diabetic mice treated with Se NPs + PRP (2:1), group 7; diabetic mice treated with Se NPs + PRP (1:2).

Histochemical observations
Hydroxyproline Healing tissues synthesize collagen, which is a component of growing cell.Hydroxyproline acts as marker of collagen that's why we evaluated hydroxyproline content in skin tissue.Concentration of hydroxyproline is a reflection and quantification of the collagen concentration.Higher hydroxyproline concentration implies faster wound healing rate.Figure 6 gives an overview of the findings.Hydroxyproline contents in groups 1 to 7 were 51.1, 12.39, 19.61, 20.90, 28.94, 30.10, and 28.96 mg/g, respectively (Figure 6).Among the diabetic groups, the maximum levels of hydroxyproline were documented in group 6 (30.10 mg/g) which implies more collagen deposition and fast healing rate.While there was a slight difference in hydroxyproline level in group 5 and group 7 (28.94mg/g and 28.96 mg/g respectively).Hydroxyproline level in group 2 decreased by 4.12 times in comparison with group 1 (normal mice).Hydroxyproline levels in group 3 and 4 were relatively elevated but still less than groups 5, 6, and 7.

Oxidative stress and antioxidant activity
Systemic oxidative stress and cellular stress response in all the experimental groups were evaluated and represented by Malondialdehyde (MDA).Antioxidant Enzymes Glutathione (GSH) level gives a reflection of the antioxidant potential of Se NPs-PRP (Fig. 6).

MDA
The lipid peroxidation status of MDA value of groups 2, 3, 4, 5, 6, and 7 was seen higher than that of group 1(normal control).While assessing the oxidative stress status, an increase in the average MDA in groups 3 and 4 was revealed compared to groups 5, 6, and 7. Group 6 and 7 showed the same MDA level.Group 2 showed the greatest value (7.94 nmol/mg).

GSH
Antioxidant activity of Se NPs-PRP were given in Fig. 6.GSH level was the greatest in group 6 (13.93 nmol/mg).The value of GSH levels in group 2 exhibited a significant decrease (to 52.86% of control levels).Groups 3 and 4 showed similar values (9.76 and 9.98 nmol/mg).However, a remarkable increase was noticed in groups 5 and 7 comparing to groups 2, 3, and 4. Findings of MDA and GSH validates the potent wound healing activity of Se NPs-PRP.As it reduced oxidative stress, and increased glutathione levels.

Discussion
This in vivo study was carried out to determine the wound healing activity of Se NPs-PRP in diabetic mice.The speed of wound healing was assessed according to the given treatment.On the 7th day, the wound area ratio was significantly small in groups 1, 5, 6, and 7 implying significant wound healing activities.Groups 3 and 4 showed moderate healing activity.Group 2 showed the lowest healing activity.On the 14th day, groups 1, 3, 4, 5, 6, and 7 appeared non-inflamed with epithelial tissues attached completely without signs of infection.Group 2 had inflamed wounds with evidence of infection.On the 21st day, the wounds of all groups were almost completely closed except group 2. Groups 5, 6, and 7 showed faster hair regrowth rate.By the 28th day, group 2 showed delayed wound closure and impaired wound contraction compared to all other groups.In Group 3, moderate healing activity is explained by PRP treatment.PRP accelerates the healing of diabetic wounds through several potential mechanisms.Their increased concentration of growth factors lowers inflammation.They also release antimicrobial peptides and serve as antibiosis agent 37 .Moreover, PRP potential antidiabetic effect though stimulated islet cell regeneration has been reported 38 .In Group 4, moderate healing activity is explained by Se NPs treatment.Se NPs treatment accelerates wound healing through several mechanisms.Their antihyperglycemic activity has been demonstrated in streptozotocin-induced diabetic rats 39 .www.nature.com/scientificreports/Se NPs with their small size and large surface area have high potential for scavenging free radicals.The oxidative stress is recognized to greatly affect the regenerative potential and cause delayed wound healing 40 .Moreover, Se NPS antimicrobial effect through disruption of bacterial cell wall is suggested to tackle bacterial resistance encountered in diabetic wounds 41 .In Groups 5, 6, and 7, the wound healing activity was significant.This implied significant synergistic effect of the Se NPs-PRP combination.Group 6 showed the highest wound healing activity with their specific ratio values.
By the end of the experiment, wound healing was observed in all histological specimens of all study groups except group 2. On the 1st day, all specimens showed acute inflammatory response as a defence mechanism against the occurred injury.This inflammation is characterized by accumulation of leukocytes within the connective tissue (injury site).On the 7th day, a layer of epithelium started to appear on the granulation tissue of all the groups except group 2. This layer is an indication of the proliferation of basal cells around the wound, and migration to the midpoint of the wound.Group 2 showed vast amounts of inflammatory cell infiltration with no granulation until week 2. Epithelialization was observed earlier in groups 1, 5, 6, and 7.At the end of the experiment period, all skin wounds healed both macroscopically and microscopically and re-epithelialized completely except group 2. www.nature.com/scientificreports/Histological scores revealed incomplete wound healing in group 2, moderate wound healing in groups 3and 4 and fast wound healing in groups 5, 6, and 7, close to that, of group 1 (normal mice).The primary indicators of wound healing include collagen formation, fibroblast proliferation, angiogenesis, and epithelialization.Group 2 showed poor collagen formation, fibroblast proliferation, angiogenesis, and epithelialization.Groups 3 and 4 showed moderate collagen formation, fibroblasts proliferation, angiogenesis, and epithelialization.Groups 5, 6, and 7 showed greater collagen formation, fibroblast proliferation, angiogenesis, and epithelialization.
The proposed mechanisms for microscopic wound healing activity are brought by PRP and Se NPs.PRP growth factors produce fibroblasts and endothelial cells which the body uses to subdue inflammation and promote healing.PRP promotes angiogenesis of wound tissue by increasing local vessel intensity and its associated (VEGF) and insulin-like growth factor 1. Furthermore, PRP promotes wound contraction and stabilizes collagen arrangement 42 .Se NPs treatment decreases apoptosis and promotes wound healing through their antioxidant and anti-inflammatory effects 43 .
These findings were in good accordance with Webster et al., who reported mild improvement of dermal fibroblast metabolic activity with adding Se NPs to electrospun silk Scaffold 44 .Also, Xu et al. indicated accelerated wound healing potential of Se NPs by reducing inflammation, enhancing granulation tissue formation, and angiogenesis.In addition to the effect of Se NPs in accelerating wound healing process we added PRP which boost the activity of Se NPs.PRP regulate the production of inflammatory cytokines interleukin-17A and interleukin-1β and increased the local vessel intensity and re-epithelialization.Besides the enhanced secretion of growth factors such as (VEGF) and (IGF-1).PRP also increases the migration and proliferation of primary cultured ESCs which accompanied by the differentiation of ESCs into adult cells following the changes of CD49f.and keratin 10 and keratin 14.PRP promotes collagen deposition and effectively reduce healing time 42 .
A healing tissue synthesizes collagen, which is a constituent of growing cells.Concentration of hydroxyproline indicates faster wound healing.Hydroxyproline levels in group 2 decreased in comparison with other groups which reflected poor wound healing.Hydroxyproline levels in groups 3 and 4 increased relatively but less than the increase in groups 5, 6, and 7.This shows the therapeutic effects of PRP and Se NPs on wound healing activity reflected in higher hydroxyproline levels.The higher levels in groups 5, 6, and 7 suggest the synergistic therapeutic effect when combined.These results are in line with Zarei et al. who reported significant increase in hydroxyproline levels with chitosan/Nano Selenium Biofilm in rats 45 .Also, Jiang indicated remarkable increase of hydroxyproline levels with chitosan-ZnO/Selenium nanoparticles scaffold 46 .Another study by Nafiu et al. demonstrated that Se and Papaya extract increased the healing potency by up-regulation of antioxidant enzymes and cyclooxygenase specific inhibition 47 .
The increase in the lipid peroxidation status and the decline in the antioxidant defence mechanisms are the main factors in the development of complications in diabetes mellitus.Excess of lipid peroxidation and reactive oxygen species (ROS) formation damage normal cells and delay wound healing 48 .For better understanding, lipoperoxidation results from the interaction of hydroxyl radicals with unsaturated fatty acids in cell membranes, leading to the formation of lipid peroxides, hydroxide radicals, and MDA 49,50 .In this study the oxidative stress status was assessed by measuring MDA and the antioxidant status by measuring GSH which protects against accumulation of free radicals in the cell 51 .Reduced GSH levels contributes to delay wound healing process 52 .Lipid peroxidation status of MDA of all groups were higher than group 1 (normal mice).Group 2 scored the highest value due to high oxidative stress.There was decrease in the average MDA in groups 3 and 4.More decrease in MDA levels in Groups 5, 6, and 7 suggesting less oxidative stress due to the therapeutic effect of Se NPs-PRP.GSH is a protective enzyme against free radical formation in tissues and it decreases in diabetic mice due to inactivation caused by generated ROS.Group 6 showed the highest GSH activity while group 2 represented the lowest GSH value (8.85 nmol/mg).Groups 3 and 4 showed similar results with values 9.76 and 9.98 nmol/mg.Significant increase was noticed in groups 5 and 7 compared to groups 2, 3, and 4. Se NPs reversed the activation of this enzymatic antioxidant, which might be due to decreased oxidative stress as evidence by decreased MDA levels too.Also, Se NPs are characterized by small size and their high surface area.This small size allows more Se atoms to scavenge a lot of free radicals.Thus, Se NPs have great scavenging activity, and boost endogenous antioxidant proteins 53 .A similar study investigated the ability of Se NPs to quench ROS and demonstrated that Se NPs inhibited lipid peroxidation in renal tissues, reflecting its ability to protect the integrity of the cell membrane 54 .It was also reported that Se NPs could elevate GSH and mRNA expression of its based enzymes (GPx and GR) in addition to (SOD) and catalase (CAT).
Based on all the obtained results, it can be concluded that Se NPs-PRP may have a potential in wound healing activity.As shown morphologically, the wound closure in diabetic mice groups treated with Se NPs-PRP (with different ratios) was very close to normal nondiabetic mice.The histological study revealed that, Se NPs could elevate the rate of proliferation, revascularization, collagen deposition and remodelling at wound sites.This agreed with the histochemical investigation of hydroxyproline which showed raised levels.Decreased MDA levels could be due to reduced oxidative stress.Increased activity of GSH may indicate the antioxidant nature of both Se NPs and PRP against oxidative stress which may have a contributory role in fasten wound healing process.
Platelet-containing plasma (PRP) has shown potential for wound healing, but it faces many challenges.First, the lack of standardization in PRP preparation leads to variation in the number of platelets that promote platelet growth, affecting the efficacy and complexity of treatment Second, although PRP provides shorter wound healing times however, the long-term benefits remain unclear, and studies suggest that treatment effects may diminish over time.In addition, PRP injections can cause side effects such as pain, inflammation, and delayed healing especially in individuals with certain underlying conditions Apart from that, the mechanisms of PRP for scarring are not understood is effectively treated is well understood, and more studies are needed to clarify the role of various tumour factors and cellular properties.Most clinical studies of PRP in wound healing are limited or lack rigorous methodologies, emphasizing the need for large, well-controlled trials to provide conclusive evidence of PRP efficacy emphasize efficiency and safety.

Conclusion
The application of nanoparticles in the treatment of diabetic wound healing is proving to be a promising and effective tool in treatment and care.Nanomaterials can stimulate several cellular and molecular processes that help with wound microenvironment through their antimicrobial, anti-inflammatory and angiogenic effect.The unique properties of selenium nanoparticles make them the therapeutic of choice in diabetic wound healing with their superior antioxidant and antimicrobial effect.Adjuvant PRP therapy can further speed the healing process in diabetic wounds with their increased concentration of growth factors.They jump start healing from inflammatory to proliferative stage.PRP triggers the body's natural healing properties, to repair and regenerate cells and tissues.The prospects of Se NPs and adjuvant PRP would be the breakthrough therapeutic in tackling the complexity of diabetic wound healing.A synergistic therapeutic effect on diabetic wound healing was demonstrated.Se NPs-PRP showed promising potential in modulating wound healing in diabetic mice.These results would have future therapeutic implications for diabetic wounds in humans.It could tackle the complexity of (DFU) and lower the incidence of lower limb amputations.

Preparation of Se NPs
Se NPs were produced by simple wet chemical method in which 0.1g of polyvinylpyrrolidone (PVP) was dissolved in 40 mL of deionized water with continuous stirring.0.6 g of ascorbic acid was added to the prepared solution while stirring for 5 min.Then 1mL of 0.96g/mL of cetyltrimethylammonium chloride (CTAC) solution was added.After 5 min of stirring 1mL of 0.25M of sodium selenite (SS) solution was added drop-wisely until the solution changed from colorless to orange 55,56 .

Preparation of PRP
Healthy group of mice was used for collecting the whole blood for PRP preparation.Whole blood (WB) by venipuncture was obtained and placed in acid citrate dextrose (ACD) tubes at room temperature then centrifuged using a soft spin.The resultant supernatant plasma containing platelets was transferred into another sterile tube (without anticoagulant) then centrifuged at a higher speed (hard) spin.The platelet-poor plasma (PPP) in upper 2/3rd of the tube was removed.The remaining PRP was in the lower 1/3rd at the bottom of the tube.

Preparation and characterization of Se NPs-PRP
4 mL of plasma was dissolved in 4 ml of deionized water and was mixed with 75 microns of the prepared nanoparticles.UV-VIS absorption spectra were obtained on Cary series UV-Vis-NIR, Australia.Transmission Electron Microscopy (TEM) images were performed on Talos F200i high resolution transmission electron microscope (Thermo-Fisher) at an accelerating voltage of 200 kV.X-ray diffraction (XRD) measurements were conducted using a Philips PW1710 X-ray diffractometer with Cu Ka radiation (k = 1.54186A˚).The XRD patterns were recorded within the 20° to 70°2Ɵ range with a step size of 0.020°2Ɵ and a collection time of 10 s per step.FT-IR spectra were recorded using a Nicolet 6700 infrared spectrophotometer to identify specific functional groups present on the surface.The charge density measurements were carried out via malvern panalytical zetasizer nano zs.

Animal ethical approval
All animal experiments were performed in accordance with the ethical approval number CU I F 42 21 from Institutional animal care and use committee (CU-IACUC) Cairo University.This study is reported in accordance with ARRIVE guidelines 57 .All experiments were performed in accordance with relevant guidelines and regulations in the National cancer institute, Cairo University.The animals were housed in polypropylene cages and allowed free access to food and water.The animals were acclimatized to standard laboratory environmental conditions, dark and light cycles (12:12 h) for 28 days.

Induction of diabetes
After 16 h of fasting, 150 mg/kg alloxan monohydrate was intraperitoneally administrated 32 .Following an alloxan injection, animals frequently exhibit increased thirst and polyuria (excessive pee) due to insulin insufficiency and hyperglycemia.As a result, the consumption of glucose water was monitored following an alloxan injection to determine how the injection affects blood sugar levels.Intake of glucose water is frequently tracked following an alloxan injection to evaluate how the injection affects blood sugar levels.Following surgery, housing arrangements for experimental mice usually entail giving them a clean, safety, cosy space with access to food and drink, as well as keeping an eye on their recuperation and general wellbeing.After five 5 days with access to food and water, the mice were screened for hyperglycemia.Mice with blood glucose greater than 200 mg/dl were considered diabetic 58 .Blood glucose was monitored using a glucometer.

Experimental design
Forty-nine male mice of the BALB/c strain aged two months with uniform weight (23 ± 0.5g) were divided into seven groups (n = 7) (Table 2).Group 1 represents untreated mice that received only food and water for 28 days.The other six groups (2-7) represent diabetic mice.

Surgical wounding
For making injuries, mice were anesthetized using 5% isoflurane.The dorsal portion of the body was shaved, and sterilized.Using a scalpel, make a cut line on each mouse (2 cm long-incision) was made.The incision was

Morphological evaluation
Photo-documentation of wound closure in all groups over 28 days was carried out.Also, Bates-Jensen Wound Assessment Tool (BWAT) was used to assess wound closure rate 33 .It is a straightforward validated wound assessment tool that allows for detailed reassessment and monitoring of healing process.This assessment tool includes 13 items such as wound size, type and depth, amount of necrotic tissue, amount of exudate, granulation tissue formation and epithelialization which documents details of the wound to improve treatment planning and re-assessment.Scores range from 1 to 5. Score 1 indicates the best healing situations while score 5 indicates impaired healing or wound deterioration 53 .

Histological evaluation
Histopathology of wounds is an essential approach to monitor healing progress, assessing morphological changes in each phase and help with diagnosis and treatment of impaired wounds [59][60][61] .Clinically, taking the biopsy at the edge of the wound makes the comparison between the ulcerated area and the surrounding skin easier.However, in the laboratory setting a biopsy for histopathological analysis should encompass the whole wound [62][63][64] .Skin biopsies were taken on days 1, 7, 14, and 21 post-surgeries.Skin samples were placed immediately into specific solutions such as 10% buffered formaldehyde and subjected to many steps of histological processing, including embedding, sectioning (4 μm formalin fixed paraffin embedded sections) and staining with hematoxylin and eosin (H&E) 65 .

Histochemical evaluation
Evaluation of collagen content through hydroxyproline: Collagen breakdown releases free hydroxyproline and its peptides.That's why hydroxyproline is used as a biochemical marker during wound healing for evaluation of tissue collagen content.It is also used as an indicator of collagen turnover after wound-healing.Increased hydroxyproline content in granulation tissue reflects increased collagen turnover, which refers to higher maturation and proliferation of collagen during wound closure 66,67 .

Hydroxyproline
To evaluate hydroxyproline, 4-mm-diameter skin biopsies were obtained after the 14th post wound day and dried in a hot-air oven at 60-70 °C.Then, all the tissue samples were hydrolyzed with 6 N HCl for 3 h at 130 °C.The hydrolyzed tissue samples were adjusted to pH 7.0 and subjected to chloramine-T oxidation.The colored adduct product formed with Ehrlich reagent at 60 °C was read at 557 nm.According to Woessner, Standard hydroxyproline was run and values reported as mg/g dry weight of tissue 68 .

Oxidative stress tests
The parameters of oxidative stress include Lipid peroxidation products (LPO) and the activity of antioxidant enzymes Glutathione (GSH).Their levels were assessed to follow up wound healing activity and the potential antioxidant effect of the nano-combination Se NPs-PRP 40 .

Malondialdehyde (MDA)
MDA is a major indicator of oxidative stress as it used for screening and monitoring lipid peroxidation induced by ROS.This assay was carried out using ab233471 Lipid Peroxidation (MDA) Assay Kit (Colorimetric).Absorbance increase was monitored at 695 nm and MDA concentration was expressed as nmol/mg protein.
Table 2. Analysis of the studied groups within the present work.

Mice groups Description
Group 1 Untreated mice (negative control)

Figure 1 .
Figure 1.TEM image of the prepared Se NPs and Se/PRP NCs (a,b).UV-VIS absorption spectra of Se NPs, PRP, and PRP/Se NPs combination (c).FRIT spectra of Se NPs, PRP, and PRP/Se NPs combination (d).XRD patterns of the prepared Se NPs and Se/PRP NCs (e).Zeta Potential Distribution of Se NPs, PRP, and PRP/Se NPs combination (f-h), respectively.

Figure 2 .
Figure 2. Representative images showing morphological changes of different experimental wound healing groups (incisional wounds on the dorsal surface in mice); a representation of follow-up during the inflammatory phase, reepithelialization and complete healing.

Figure 3 .
Figure 3.The wound area concerning different days (the rate of wound closure over time).

Table 2
and poor epithelialization.The histological scores revealed incomplete wound healing in group 2, and moderate wound healing in groups 3 and 4. Groups 5, 6, and 7 revealed a fast wound healing rate approximately equal to the normal rate of group 1 (normal mice) as shown in Table1.From these histological results, Se NPs-PRP is shown to enhance wound granulation tissue formation and epithelialization to accelerate wound closure.

Table 1 .
Histopathological assessment of wound healing parameters.+ + + HIGH presence of extracellular matrix, + + MODERATE presence of extracellular matrix and + DISCRETE incomplete presence of extracellular matrix.
Vol:.(1234567890) Scientific Reports | (2024) 14:5346 | https://doi.org/10.1038/s41598-024-54064-2 made paravertebral and perpendicular to the skin cleavage line.All wounded mice were photo-documented to evaluate the healing rate clinically.Following surgical procedures, housing arrangements for experimental mice usually entail giving them a clean, safety, adequate space with access to food and drink, as well as keeping an eye on their recuperation and general wellbeing.The analgesic (Ketoprofen) was given intramuscularly (IM) after surgery to alleviate pain till the symptoms were resolved.